C. Y. Kuo, J. C. Alexander, J. H. Lumsden and R. G. Thomson'. ABSTRACT. Safety evaluations of two thermotolerant filamentous fungi, Cephalosporium eichhor ...
Subchronic Toxicity Test for Two Thermotolerant Filamentous Fungi Used for Single Cell Protein Production C. Y. Kuo, J. C. Alexander, J. H. Lumsden and R. G. Thomson' ABSTRACT
Safety evaluations of two thermotolerant filamentous fungi, Cephalosporium eichhorniae 152 (C. 152) and Rhizopus chinensis 180 (R. 180), grown on a sugar-salts medium were carried out through feeding the biomasses to rats at 20% or 40% dietary levels for 90 days. There was a control group fed soybean meal. Weight gain and feed consumption for rats fed 20% C. 152 were equal to those for the control animals, but were depressed in the other three groups, especially the rats fed R. 180. All animals appeared normal and healthy, except that transient alopecia was xound for a short duration in the fungi-treated rats in the initial period. The cause of this lesion is not clear. At the end of the feeding trial, clinical determinations of constituents in blood and urine samples were conducted. The animals were autopsied and weights for four organs were taken. Histopathological examinations for 26 different tissues were carried out. Mild changes were found in both C. 152 and R. 180-treated rats but most of these were not considered to be related to treatment.
RASUMA Les auteurs ont verifie l'innocuite de deux champignons thermotolerants: Cephalosporium eichhorniae 152 (C. 152) et Rhizopus chinensis 180 (R. 180), qu'ils avaient cultives sur un milieu a base de sucre et de sels et ensuite pulverises. A cette fin, ils donnerent a des rats, durant 90 jours, une moulee qui contenait 20% ou 40% de champignons pulverises. Le groupe de rats temoins ne
regut
que du
'Department of Nutrition (Kuo and Alexander) and Department of Pathology (Lumaden and Thomson), University of Guelph, Guelph, Ontario NlG 2W1. Submitted July 24, 1978.
so
tourteau de soya. Le gain de poids et la consommation alimentaire des rats dont la moulee contenait 20% de C. 152 s'avererent egaux a ceux des temoins; ils manifesterent cependant une baisse, chez les sujets de tous les autres groupes, particulierement chez les rats qui recevaient la poussiere du champignon R. 180. Tous les rats semblaient normaux et sains; les sujets experimentaux developperent cependant, au debut de l'experience, une alopecie transitoire d'etiologie indeterminee. Vers la fin de l'experience, les auteurs determin'e. rent certains parametres hematologiques et urinaires. Ils effectuerent aussi la necropsie des rats, la pesee de quatre de leurs organes et l'histopathologie de 26 de leurs tissus. Les lesions benignes des tissus des rats experimentaux ne semblerent pas attribuables a
l'experience.
INTRODUCTION The world protein shortage together with the waste disposal problem has greatly stimulated interest and research in the production of single-cell protein (SCP). Over the years, a considerable amount of work has been devoted to the development and production of SCP from different microorganisms grown on various substrates with the aim of providing protein for animal or human nutrition (5, 22). In the present studies, a simple, low cost, nonaseptic process for the conversion of cassava, a starchy tropical root crop, into fungal protein for use as animal feed was sought. The details were given in preliminary reports by Reade and Gregory (17). Upon the second search for adequate cultures, two thermotolerant filamentous fungi, Cephalosporium eichhorniae 152 (C. 152) and Rhizopus chinensis 180 (R. 180)
Can. J. comp. Med.
were found to have the highest protein efficiency ratios (PER) among seven potential cultures selected (13). Further nutritional evaluations were conducted and the results will be published elsewhere. this report describes subchronic safety evaluations of the two fungi, as it is recognized that a newly developed protein source must be tested thoroughly to ensure the safety of its use (6).
MATERIALS AND METHODS PRODUCTION OF FUNGAL BIOMASSES
Thermotolerant filamentous fungi Cephalosporium eichhorniae 152 (C. 152) and Rhizopus chinensis 180 (R. 180) were grown on a sugar-salts medium composed of the following in g/l: sugar (glucose for C. 152 and sucrose for R. 180), 40.0; KH2PO4, 2.0; MgSO4*7H20, 0.2; KC1, 0.1; FeSO4-7H20, 0.02; ZnSO4*7H20, 0.02; CaC12S2H20, 0.02; (NH4) 2SO4, 9.0; CO (NH2) 2, 2.0; corn steep liquor (sterilized at 121°C for 30 min), 30 ml//. Fermentations were carried out in a 120 / selfaspirating fermentor (3). Incubation was at 47°C and pH 4.5 for 18-24 hr. The fungal biomasses were harvested by filtering them through laundry bags and they were washed once with tap water. All cells were freezedried and ground to powder. Tests for six mycotoxins (aflatoxins, citrinin, ochratoxin, zearalenone, T-2 and sterigmatocystin) were carried out' before these biomasses were fed to rats and negative results were obtained. RATS AND DIETS
Male weanling rats of the Wistar strain, weight range from 45 to 55 g, were obtained from Woodlyn Laboratories, Guelph, Ontario. They were randomly divided into five groups of ten with the average weight approximately the same and fed diets containing 0%, 20% or 40% of fungal biomass C. 152 or R. 180 for 90 days. The composition of the basal diet is given in Table I. Protein was provided at a 20% dietary 'Laboratory of Toxicology, Veterinary Service Laboratory of Ontario Ministry of Agriculture and Food, University of Guelph, Guelph, Ontario.
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level at the expense of corn starch. Soybean meal (SBM) was used as the sole protein source in the control diet and was added to each fungal diet in sufficient quantity to give a 20% final dietary protein level, as indicated in Table I. Supplementation with DL-methionine was carried out to give a level of 0.7% methionine in each diet. The rats were fed and watered ad libitum. Weight gains and feed consumption were recorded weekly and the animals were observed for any changes in normal appearance.
CLINICAL DETERMINATIONS On the 86th day of the feeding trial, rats were deprived of water for 24 hr. During the terminal 12 hr of this period, urine was
collected and subjected to osmolality and specific gravity determinations to evaluate renal tubular concentrating ability. Osmolality was determined with an Advance Osmometer (Model 3D).2 Specific gravity was measured using a TS Refractometer2 (American Optical Company). At the end of the 90-day feeding trial, urine samples from individual rats were collected for 24 hr. The color and clarity of each sample were recorded and osmolality and specific gravity were determined as described above. The following urine analyses were carried out based on turbidity or color comparison, as described by Kark et al (14): protein (20% sulphosalicylic acid),' pH (Dual-Tint pH papers3), blood or free hemoglobin (Ames Hematest tablets3), ketone bodies (Ames Acetest tablets3), sugar (Ames Clintest tablets3) and bilirubin (Ames Ictotest tablets3). Urine sediment obtained by centrifugation of each sample at 2,500 rpm for one min was stained with Clay Adams Sedi-Stain.3 Microscopic examinations were carried out for the presence of cells, casts, crystals, bacteria, sperm, mucous thread and fat. At the termination of the feeding study, the animals were fasted overnight. Nine hours after the fasting began, individual rats were lightly anesthetized by ether. Approximately 2 ml of blood was collected from each rat using the retro-orbital bleed2Canadian Laboratory Supplies, Toronto, Ontario. 3Fisher Scientific Company Ltd., Toronto, Ontario. 4When positive findings were obtained, Ames Bili-Labstix Test was carried out as a check of the result.
51
TABLE I. Composition of Diet for the Subchronic Toxicity Test Dietary Ingredients (%) Corn Vitamin Salt Corn Soybean C. 152 R. 180 Starch" OUlb Mixture" Premild Alfaflocb Meal 1.0 1.0 4.0 45.9 6.0 41.7 1.0 1.0 4.0 45.5 6.0 20.0 22.0 1.0 1.0 40.0 45.1 4.0 6.0 2.3 1.0 20.0 43.4 4.0 6.0 24.2 1.0 1.0 40.0 41.0 4.0 1.0 6.0 6.7 &St. Lawrence Starch Co. Ltd., Port Credit, Ontario bCanada Starch Co., Scarborough, Ontario -Salt Mixture XVII, ICN Pharmaceuticals, Inc., Cleveland, Ohio dPrepared from the following vitamins in g/kg mixture; vitamin A acetate (500,000 I.U./g) 0.6, vitamin D3 (400,000 I.U./g) 0.4, dl-a-tocopheryl acetate (500 I.U./g) 10.0, menadione 0.02, choline chloride 150.0, niacin 3.0, calcium pantothenate 2.0, riboflavin 0.5, thiamin HCl 0.3, vitamin B12 (0.1% trituration of cyanocobalamin with mannitol) 1.0, pyridoxine HCl 1.5, folic acid 0.2, d-biotin 0.02, inositol (dihydrate) 10.0. Celluflour added to 1 kg (American Dietetic Supply Inc., La Grange, Illinois)
Diet Soybean meal 20% C. 152 40% C. 152 20% R. 180 40% R. 180
ing technique (20). These blood samples were collected in 2.5 ml EDTA-tubes and taken immediately for hematology and plasma glucose analyses. Total white blood cells (WBC), total red blood cells (RBC) and hemoglobin (Hgb) were obtained directly with a Coulter counter (Model S5) and hematocrit (Hct) was determined with an Adams microcentrifuge.6 Mean cell volume (MCV), mean cell homoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated from the above values. White cell differentiations were carried out on 100 leukocytes according to the method described in Schalm (18) and absolute values were calculated as a percentage of the total leukocyte counts. All data, except those for eosinophils, were converted to normal distribution by square root transformation and then subjected to analyses of variance. In the case of eosinophil counts, the Kruskal-Wallis nonparametric analysis of variance was used to analyze the data because all transformation methods failed to convert the data into a normal distribution (19). The percentage of reticulocytes in terms of red cells was determined, using the method of Brecher and Schneiderman (6). Blood also was collected from each rat by heart puncture. These blood samples were allowed to clot at room temperature for one hr, and spun twice at 3,000 rpm for ten min to obtain serum samples, which were subjected to the following analyses: serum alkaline phosphatase activity by the method of Babson et al (4), serum gluta-
mate-oxaloacetate transaminase (SGOT) and serum glutamate-pyruvate transaminase (SGPT) activities using an LKB 8600 Reaction Rate Analyzer,7 blood urea nitrogen (BUN) by the method of Crocker (7), blood glucose by the method of Feteris (11) and total serum protein using the biuret method (2). Serum proteins were separated through electrophoresis at pH 8.6 for 42 min, in a Corning-ACI Agarose Film and Cassette electrophoresis system.' For all clinical determinations, the results were evaluated statistically by Tukey's
5Coulter Electronics of Canada Ltd., Mississauga, Ontario. 6Fisher Scientific Company Ltd., Toronto, Ontario.
7Boehringer Mannehein, Quebec, Canada. 8Fisher Scientific Company Ltd., Toronto, Ontario.
52
multiple comparison procedure (19). ORGAN WEIGHTS AND HISTOPATHOLOGICAL EVALUATIONS
After blood was collected for clinical determinations, the rats were killed by ether and dissected. Thyroid glands, adrenal glands, kidneys and livers were removed and weighed. These organs, together with the following tissues were dissected and selected parts were trimmed to a width of 3 mm and fixed immediately in 10% buffered formalin for histopathological examinations: salivary gland, cervical lymph node, trachea, thymus, heart, lung, pancreas, spleen, stomach, mesenteric lymph node, duodenum, ileum, cecum, urinary bladder, prostate, seminal vesicle, testis, sciatic nerve, femur, muscle, skin, spinal cord, brain and pituitary gland. After fixation, bone sections were decalcified, then, together with all other sections were
Can. J. comp. Med.
subjected to a routine process of dehydration and paraffin embedding. Slides were made from the paraffin block and were stained with hematoxylin and eosin. Various types of lesions found in the selected tissue section under a light microscope were graded on a numerical scale to produce scores from zero to three as to the severity of the lesions. Lesions were summarized on an organ and treatment group basis for comparisons to be made. The criteria used for the main microscopic lesions are described briefly below: Respiratory system - the size and distribution of the lymphocytic cuffs in the mucosa of the trachea, or around the bronchioles and blood vessels of the lung. Stomach - the presence of eosinophils in the mucosa in the region of the junction of glandular and squamous mucosa, also, hyperplasia, vacuolation and hyperkaratosis of the squamous epithelium in this region was evaluated. Bladder - the existence of proteinaceous material in the lumen and the thickening of the epithelium. Kidney - the existence of syncytial cells in renal tubular and lymphocytic foci in the tissue, deposition of granular and protein casts in the tubules. Pancreas - the relative granulation of the acinar cells and the mononuclear cellular accumulation. Nerve system - the size and distribution of vacuoles in the tissues. Reproductive system the existence of cells in the lumen of seminal vesicles or the seminiferous tubules of testes and the hypertrophy or atrophy of the epithelial cells of the prostate. Thyroid - the existence of cells and debris in the lumen of the acini. The appearance of lymphocytic foci in various tissue sections also was recorded.
RESULTS FEEDINGS STUDIES
Average weight gain and feed consumption of each group of rats were recorded at
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four, eight and twelve weeks, and the feed efficiency was calculated (Table II). The performance of the 20% C. 152 group was equal to that of the control group, while the other three fungal groups had lower weight gains and feed consumption. A dosedependent decrease in weight gain and feed intake also was found in all fungal groups. The rats in each group appeared healthy and normal with one exception; some transient alopecia was found in the fungitreated animals. It followed a particular pattern in that hair was partially lost in a distinct area of approximately 2.5 cm diameter above the eyes and around the neck. The lesion appeared two weeks after the rats had been put on the experimental diets and disappeared less then two weeks later.
CLINICAL DETERMINATIONS Values obtained for the renal tubular concentrating test and urinalyses are shown in Table III. No significant difference was found among the five groups for osmolatity and specific gravity values. A dose-dependent decrease of urinary pH was found in the fungi-treated groups when compared to the control group. Small amounts of protein were found in most of the urine samples. However, there was no significant difference among the five groups including the control. None of the other chemical analyses and microscopic examinations of urine samples revealed significant findings. Hematological values are shown in Tables IV and V. Small changes were found in the values for MCV, percentage of reticulocytes and absolute counts for band cells. Average values for MCV from the 40% R. 180treated group was slightly but significantly higher than that for 40% C. 152-treated group but both were not significantly different from that for the control group. Rats fed 20% R. 180-diet had a reticulocyte value significantly lower than that for the control. The absolute band cell counts for 40% R. 180-treated rats were significantly lower than those for 40% C. 152-treated rats but no significant difference was found between the control and any of the fungal groups. Values for blood biochemical and enzyme analyses are shown in Table VI. Serum alkaline phosphatase activity was elevated in 40% C. 152-treated rats when compared
53
TABLE II. Weight Gain, Feed Consumption and Feed Utilization of Rats in the Subchronic Toxicity Testab Feed Consumption Feed Efficiency' (g/rat/day) Body Weight Gain (g) 4wk 8 wk 12 wk 4wk 8 wk 12 wk 4wk 8 wk 12 wk Diet 18.3' 17.3' 15.1' 3.1' 3.9 308.81,2 392.5' 2.51 170.0' Soybean meal 18.4' 2.4' 3.1 401.3' 15.6' 17.6' 3.91 182.1' 321.5' 20% C. 152 13.32 15.52 16.22 4.01 2.71.2 3.1' 138.92 280.82 347.02 40% C. 152 3.42 4.21,2 11.33 13.33 14.73 2.92 294.83 109.33 216.73 20% R. 180 3.83 8.54 10.14 3.43 4.32 11.64 224.94 149.14 17.34 40% R. 180 *Mean for ten rats in each group bValues with different superscripts in the same column are significantly different (P = 0.05) by Tukey's multiple comparison procedure, (19) 'Feed Efficiency = Total feed consumption (g)/Total body weight gain (g) TABLE III. Values for Renal Tubular Concentration Test and Urine Analysesa,b Renal Tubular Concentration Test Osmolality Specific Diet Gravity (mOsm/kg) Soybean meal 2929 i 82 1.079 i 0.002 20% C. 152 3139 + 175 1.082 + 0.004 40% C. 152 2792 ± 205 1.076 -fi 0.004 20% R. 180 3167 ± 62 1.085 ± 0.002 40% R. 180 3135 ± 130 1.084 i 0.003 -Mean ± S.E. for ten rats in each group bValues with different superscripts in the same multiple comparison procedure (19)
Osmolality (mOsm/kg) 1136 + 89 954 + 80 963 + 142 1033 ± 156 1253 -+- 93
Urine Analyses Specific pH Gravity 7.6 i 1.031 i 0.002 1.026 + 0.002 6.4 ± 5.9 ± 1.027 i 0.004 6.5 ± 1.029 ± 0.004 1.035 ± 0.003 5.9 ±
column are significantly different (P
=
0.111 0.082 0.043 0.092 0.033
Protein (mg/dl) 45 ± 20 51 i 12 47 ± 20 50 ± 14 45 ± 14
0.05) by Turkey's
TABLE IV. Erythrocyte Dataa,b.o
Diet
Red Blood Cells
(106/4l)
Packed Cell Volume (%) 48.0 48.7 49.0 48.6 50.4
Mean Corpuscular Volume (v3) 571,2 571,2
Hemoglobin (g/dl) 16.1 16.7 16.8 16.5 16.6
Mean Cell HemoMean Cell globin HemoConcen- Reticuglobin tration locytes (%) (S) (pg) 19.0 33.5 4.71 34.4 3,61.2 19.6 34.4 3.81,2 19.1 2.02 34.0 19.9 19.8 33.0 2.91.2
8.42 Soybean meal 8.51 20% C. 152 56' 8.78 40% C. 152 581.2 8.33 20% R. 180 602 8.42 40% R. 180 -Mean for ten rats in each group bValues with different superscripts in the same column are significantly different (P = 0.05) by Tukey's multiple comparison procedure (19) ,All data were converted by square root transformation before statistics were applied. For data smaller than ten, the formula (datum + 0.5)0-5 was used for transformation
to that for the control animals. The average SGPT activity for 40% R. 180-treated rats was significantly higher than that for 20% C. 152-treated rats but both values were not significantly different from the control. The total serum protein level was decreased in all fungal groups and 40% R. 180-treated rats had a serum albumin level significantly lower than that for the control group.
54
ORGAN WEIGHTS Rats fed diets containing R. 180 had relatively heavy thyroids and livers (Table VII). Dose-dependent enlargements of adrenals and kidneys were found in fungitreated rats, with the effect greater for R. 180-fed animals.
Can. J. comp. Med.
TABLE V. Leukocyte Data (cells/p.j)&.b Total White Blood Segmented Band Cells Diet Cells Eosinophils Lymphocytes Monocytes Neutrophils 9820 520 7720 1170 Soybean meal 901,2 30 10470 8100 560 1370 951.2 20% C. 152 53 11200 40% C. 152 8670 630 1350 1341 146 9850 7340 390 1550 1231,2 20% R. 180 168 182 8840 420 1540 6540 40% R. 180 51 aMean for ten rats in each group bValues with different superscripts in the same column are significantly different (P = 0.05) by Tukey's multiple comparison procedure (19) cAll data except for those for eosinophils were converted by square root transformation before statistics were applied. Eosinophil data were analyzed by nonparametric one-way analysis of variance (19)
HISTOPATHOLOGICAL EVALUATIONS
Histopathological lesions were evaluated by visual observation without measurements and the results are recorded in Table VIII as histopathological scores. For the organs not included in the table, microscopic examinations also were carried out but no lesions were observed. There were no distinct differences among the scores for the five groups. However, in the case of the alimentary system, there was a trend that rats fed diets containing R. 180 had higher histopathological scores than those fed C. 152 or the control diet.
DISCUSSION The lower weight gains of the R. 180treated rats were closely related to decreased feed intake. The average feed consumption of R. 180-treated rats at 12 weeks was nearly 30% less than that for the control rats and 24% less than that for the C. 152-treated rats. When the feed efficiency was calculated, there was only a 6.4% difference between the R. 180 diets and control, or the C. 152 diets. The decreased feed intake could have been due to the dusty and bulky nature of the fungal biomasses, with R. 180 more affected than C. 152. Similarly, rats fed the higher level of fungal diets were receiving dustier and bulkier rations than those fed less fungal material which might have affected their feed intake and growth. Duthie (10) fed rats with different levels of Fusarium graminearum in the diets and reported
Volume 43 - January, 1979
similar dose-dependent decreases in weight gain and feed intake. The cause of the transient alopecia is not clear. Cases of infectious alopecia by fungus Trichophyton mentagrophytes on rats of the Wistar strain have been reported (9). However, the lesions occurred at a different location (around the base of the tail) and at a different age of the rats (two to four months old). This lesion is not likely to be an infectious effect of R. 180 or C. 152 because the two fungi belong to different genera. It is recognized that a tryptophan or a zinc deficiency can cause alopecia. However, chemical analyses revealed that adequate amounts of both tryptophan and zinc were present in the diets. Urine concentration as measured by osmolality and specific gravity indicated that rats in all groups had normal renal tubular concentrating ability and were capable of concentrating their urine when deprived of water, as expected for healthy rats. The dose-dependent decrease of urinary pH for fungi-treated rats might have been due to contamination of the urine samples by spilled acidic feed, as both C. 152 and R. 180 were produced in an acidic medium and the biomasses incorporated in each fungal diet were acidic. The spilled feed also might have been partially responsible for the nontreatment-related proteinuria, which was indicated by both the turbidometric procedure and Ames Bili-Labstix test. The lower reticulocyte value in 20% R. 180-treated rats was minimal and was not considered to be clinically important because other red cell parameters indicated that adequate production was present. Although small differences in absolute
55
band cell numbers occurred between groups, of the groups had a clinically significant increase in band cells. The elevation of serum alkaline phosphatase activity in 40% C. 152-treated rats C was not accompanied by any histopathological lesions in the liver. Khor et al (15) fed rats fungal diets, de Groot (8) fed rats nX +++++ _ yeast diets and both found elevated serum 4) alkaline phosphatase levels in the animals. o'9 C6¢ncscaian r'i C'The mild decrease of total serum protein and serum albumin from R. 180 and 40% : - et C. 152-treated rats probably were due to absorption of dietary constituents. As E---i 6poor 0 :3 10 previously discussed, feed intake by each H H H ~of these three groups of rats was depressed. n sD 41 8 This might have caused certain degrees of C dietary deficiencies and consequently in_ > > csJ* terfered with liver protein synthesis. This . 66666 E E o o o deficiency or imbalance also might oodietary H H -H lEH have stressed the animals and caused an etl 0X increased release of ACTH with hyperOm 00 s e n u, s e > plasia and enlargement of the adrenal &4 Hglands, as indicated in Table VII. Volesky " ", N nand his co-workers in fungi feeding studies .o with rats also found a dose-dependent en--4 c, largement of the adrenal glands (23). ~o cq Q OOdose-dependent tThe t enlargement of kid_ _-_ _ _ neys from fungi-treated rats could have been due to renal tubular hypertrophy in response to the high dietary protein and HDM O ncq ,2 o o cs0_ -nonprotein-nitrogen levels. Enlargement of D -H -H -H -H -H kidneys has been shown with rats fed high levels of microbial protein of yeast, bac_ m cq c i terial or fungal origin (1, 15, 21) and some researchers attributed this enlargement to " E-^ Lno_rs the high dietary protein level (8, 21). -H H+ -H -fl . O
.5
oo999
none
n
E~
ecs_clo
'-4
X
GO
a0 0 O OO
X
E
= X oo -_ 00 wo0 V tlO8
o+m --H-H-H> o-o
Most of the histopathological lesions ob-
served were common to animals of all groups and some of the changes were due to the fact that the rats were not specific free. There was no clear evidence pathogen of any lesion being related to a specific diet. The gastric lesions were highly sug-
gestive of parasitic infection because of X66 ° °s -H --H-H X + + + of eosinophils and the thicke Xthe presence mg of the epithelium at the squamous . 0000 thelial cells. These were common to all o to .0 LO
o
u
56
Can. J. comp. Med.
TABLE VII. Organ Weightss.bo Adrenal Liver Thyroid Kidney Diet (mg/1OOg body wt) (mg/1OOg body wt) (g/lOOg body wt) (g/lOOg body wt) 3.8 + 0.412 8.2 0.53 + 0.011 2.81 0.51 0.061,2 Soybean meal 7.6 1 0.41 3.4 + 0.41 0.54 i 0.021.2 2.60 d 0.061 20% C. 152 4 9.3 ± 0.51.2 0.59 :i: 0.022 2.70 4.3 0.051 0.31,2 40% C. 152 9.0 + 0.71,2 0.61 1 0.012 4.8 ± 0.7l,2 2.99 I 0.082 20% R. 180 0 11.0 j 0.Q72 0.69 i 0.023 2.99 : 0.052 5.5 .72 40% R. 180 sAll organ values were fresh weight, and for kidney and adrenal they were the combined weight of the left and right organs bMean S.E. for ten rats in each group ,Values without a common superscript in the same column are significantly different (P = 0.05) by Tukey's
multiple comparison procedure (19)
TABLE VIII. Evaluation of Histopathological Lesions
System or Organ ReRespiratory productive Alimentary Nervous Diet System System System System Kidney Liver Pancreas Heart Total 25 37 32 15 1 0 166 7 49 Soybean meal 14 4 20% C. 152 29 60 33 45 13 0 198 35 14 25 10 3 4 57 39 187 40% C. 152 42 4 20% R. 180 26 59 52 15 15 4 217 51 64 34 15 0 0 0 23 40% R. 180 187 61 12 8 128 188 57 225 955 Total 276 *Total scores for ten rats in each group
groups but were more numerous in the fungi-treated animals. Such lesions are often incidental findings in rats but this organ should be closely examined in future
studies. Although some mild changes were found in both clinical and histopathological examinations, negative findings were obtained in most of the extensive evaluations. All animals appeared healthy throughout the feeding trial and no overt changes except for transient alopecia were observed. Thus, the experimental diets containing 20% or 40% C. 152 or R. 180 were not considered to be injurious to the rats when fed for 90 days.
ACKNOWLEDGMENTS We wish to thank Dr. K. F. Gregory for consultation and assistance in preparing the mold samples. This work was carried out with the aid of a grant from the International Development Research Centre, Ottawa.
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January, 1979
REFERENCES
1. AGREN, G., U. STENRAM, H. NORDGREN, A. EKLUND and J. A. GLAS. Some biological effects observed in rats fed on single cell proteins of yeast and bacterial origin. Nutr. Metabol. 17: 20-36. 1974. 2. A.O.A.C. Official Methods of Analysis, 10th Edition. Washington, D.C.: Association of Official Agricultural Chemists. 1965. 3. AZI, F. A., A. G. MEIERING, C. L. DUITSCHAEVER and A. E. READE. Air injection by self-aspirating impeller in aerobic fermentation. J. Milk Fd Technol. 38: 94-99. 1975 4. BABSON, L., S. J. GREELEY, C. M. COLEMAN and G. E. PHILLIPS. Phenolphthalein monophosphate as a substrate for serum alkaline phosphatase. Clin. Chem. 12: 482-490. 1966. 5. BIRCH, G. G., K. T. PARKER and J. T. WORGAN. Food from Waste. New York: Applied Science Publishers. 1976. 6. BRECHER, G. and M. SCHNEIDERMAN. A time saving device for the counting of reticulocytes. Am. J. clin. Path. 20: 1079-1083. 1950. 7. CROCKER, C. L. Rapid determination of urea nitrogen in serum or plasma without deproteinization. Am. J. med. Technol. 33: 361-365. 1967. 8. DEGROOT, A. P. Safety evaluation studies with SCP. PAG Bull. 5(3): 41-44. 1976. 9. DOLAN, M. M., A. M. KLIGMAN, P. G. KOBYLINSKI and M. A. MOTSARAGE. Ringworm epizootics in laboratory mice and rats: experimental and accidental transmission of infection. J. invest. Derm. 30: 23-25. 1958. 10. DUTHIE, I. F. Animal feeding trials with microfungal protein. In Single-Cell Protein. Part II. S. R. Tannenbaum and D. I. C. Wang, Editors. pp. 605544. Cambridge, Massachusetts: The MIT Press. 1975. 11. FETERIS, W. A. A serum glucose method without protein precipitation. Am. J. med. Technol. 31: 17-21. 1965. 12. FLYNN, R. J. Parasites of Laboratory Animals. Ames, Iowa: The Iowa State University Press. 1973. 13. GREGORY, K. F., A. E. READE, J. SANTOSNUNEZ, J. C. ALEXANDER, R. E. SMITH and
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17.
S. J. MacLEAN. Further thermotolerant fungi for the conversion of cassava starch to protein. Anim. Feed Sci. Technol. 2: 7-19. 1977. KARK, M. R., J. R. LAWRENCE, V. E. POLLARK, C. I. PIRANT, R. C. MUEHRCKE and H. SILIVA. A Primer of Urinalyses. New York: Harper and Row. 1970. KHOR, G. L., J. C. ALEXANDER, J. H. LUMSDEN and G. J. LOSOS. Safety evaluation of Aspergillus fumigatus grown on cassava for use as an animal feed. Can. J. comp. Med. 41: 428-434. 1977. PROTEIN ADVISORY GROUP. Preclinical testing of novel sources of protein. United Nations, New York. PAG Guideline No. 6. 1972. READE, A. E. and K. F. GREGORY. High-temperature production of protein-enriched feed from cassava by fungi. Appl. Microbiol. 30: 897-904. 1975.
18. SCHALM, 0. W., N. C. JAIN and E. J. CAROLL. Veterinary Hematology. 3rd Edition. Philadelphia, Pennsylvania: Lea and Febiger. 1975. 19. STEEL, R. G. D. and J. H. TORRIE. Principles and Procedures of Statistics. Toronto, Ontario: McGrawHill Book Company. 1960. 20. STONE, S. H. Method for obtaining venous blood from the orbital sinus of the rat or mouse. Science 119: 100. 1954. 21. STRINGER, D. A. and A. B. WILSON. Toxicological studies with "Pruteen". PAG Bull. 6(3): 45-49. 1976. 22. TANNENBAUM, S. R. and D. I. C. WANG. SingleCell Protein. Cambridge, Massachusetts: The MIT Press. 1975. 23. VOLESKEY, B., J. E. ZAJIC and K. K. CARROLL. Feeding studies in rats with high protein fungus grown on natural gas. J. Nutr. 105: 311-316. 1975.
BOOK REVIEW
INFECTION AND PREGNANCY. Edited by C. R. Coid. Published by Academic Press, London. 1977. 599 pages. Price $41.00.
"Infections which influence the establishment and continuation of a healthy pregnancy and the subsequent well-being of the young are of considerable interest and importance in both human and veterinary medicine." "The object of this volume is to bring together various topics considered to be important in the field of comparative medicine." The editor has done a commendable job of integrating the contributions of a number of authors into a volume which is one of the few to stress the comparative aspect of the subject. The book is divided into fourteen chapters, each intended to be self-contained. This has resulted in some overlap of material as well as some fragmentation of information on many disease
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entities. However, this is a minor criticism and does not seriously detract from the readability of the book. The first three chapters are reviews of the main aspects of the subject in man, in farm animals and in laboratory animals. In the fourth chapter infections and infertility (problems in the establishment of pregnancy) are considered in man and animals. The remaining ten chapters cover in more detail specific topics related to the various consequences of maternal infections on fetal and postnatal development. The chapters are well organized, commencing with a short table of contents and concluding with an excellent list of references. The book should be a valuable addition to the libraries of graduate students and research workers in microbiology, pathology and theriogenology as well as practitioners who have an interest in the comparative aspects of infections and pregnancy. - P. A. Doig.
Can. J. comp. Med.
